An understanding of microbial evolution is seen as increasingly important to an understanding of disease and the design of effective therapies. As such, this proposal aims to investigate the genetic, biochemical and physiological mechanisms underpinning evolution in microbes. Of particular interest is an assessment of the degree to which adaptive evolution is predictable. For if evolution is predictable, more efficacious therapies might be designed, undesirable evolutionary outcomes (e.g. multi-drug resistance) avoided, the origin and evolution of epidemics better understood, and as a consequence better strategies in public health implemented. An understanding of microbial evolution remains elusive because studies are either overwhelmed by complexity or reduced to the trivially predictable. However, the lactose pathway of E. coli represents a unique balance between complexity and simplicity. It is sufficiently complex that a diversity of adaptive responses - specialists, generalists, commensals - arise, yet it is sufficiently simple that each can be analyzed in detail. This balance between complexity and simplicity enables the predictability of adaptive evolution to be investigated. Strong frequency dependent selection maintains variation in lactose operons of E. coli during competition for limiting mixtures of galactosides. Long-term chemostat competition experiments reveal that new adaptations may intensify the frequency dependence or they may diminish it to the point whereby the polymorphism is lost. Balanced polymorphisms can arise from within a single clone. The evolution of specialists towards particular galactosides promotes polymorphism and suggests that trade-offs at the molecular level govern the evolution of diversity. The evolution of generalists (strains capable of efficiently metabolizing all galactosides) destabilizes polymorphisms and demonstrates that trade-offs are not inevitable. The genetic, physiological and biochemical mechanisms that promote the appearance, and loss, of these polymorphisms will be determined. Replicate experiments will determine the predictability of evolutionary outcomes. Evolved strains will be analyzed to determine the population level interactions that result in stabilizing or destabilizing the polymorphisms. Biochemical studies will determine the physiological basis for the evolution of specialists and generalists, and to determine the molecular causes of trade-oils. Mutational changes will be sequenced to determine the predictably of evolution at the molecular level.